Stanford Microfluidics Laboratory

Microsecond Mixing for Investigation of Protein Folding-in collaboration with LawrenceLivermore National
Laboratory

MotivationImportant
structural events in protein folding are known to occur on sub-millisecond
time scales, but studies of fast protein folding kinetics have been limited
by mixing rates of protein and denaturant solutions. Simple proteins often
have folding rates on the order of 10 microseconds, whereas fast mixing
experiments have yet to probe these short time scales.

Project
DescriptionWe have
developed a diffusion-based microfluidic mixer to investigate protein
folding on a microsecond time scale. Our mixer uses hydrodynamic focusing
of pressure driven flow to create a sub-micron wide stream of protein and
denaturant solutions. The denaturant diffuses out of this thin sheet faster
than the protein and the proteins see a local drop in denaturant concentration
causing them to fold.The mixer
geometry and flow conditions are optimized with numerical simulations, and
we use micro-PIV to visualize velocity fields in the mixer.

Figure 1. Schematic of mixer and SEM of silicon device. A)
Red is denatured protein solution, blue is buffer. A solution of denatured
proteins is injected from the north channel and hydrodynamically focused into a thin sheet with buffer from the side channels. The
denaturant diffuses out of the focused stream inducing the proteins to
fold. The folding kinetics are then optically
detected downstream.

Mixer optimizations

Our mixers use over eight orders
of magnitude less labeled protein sample mass flow than a previously
reported ultra-fast protein folding mixer, with flow rates of 3 nl/s and protein concentrations of tens of nanomolar.We have demonstrated the fastest mixers of any kind, and can mix two
streams in a few microseconds.Our work includes the design and optimization of mixers using models
of convective diffusion phenomena; and a characterization of the mixer
performance using micro-particle image velocimetry, dye quenching, and
Förster resonance energy transfer (FRET) measurements of
single-stranded DNA.

Figure 2.Simulations of flow field in mixer including
velocity field and convective diffusion of denaturant molecules from the
center stream to the outer, sheath streams (on the left).These simulations have been
validated using micro-PIV experiments (Hertzog,
2004).On the right is an
oblique view of streamlines at relatively high Reynolds numbers (Re = 30),
where flow becomes strongly three-dimensional and centripetal flow effects
lead to dispersion and harder-to-control mixing.

Figure
3. An example mPIV velocity field
obtained in the actual mixer and used to validate the model predictions and
optimization process. (Image inverted for clarity). Maximum measured velocity here was 1.8
m/s.

We
have performed optimizations of mixer geometry and flow conditions
(particular center-to-side flow rate ratio).Our optimization calculations
resulted in two important counter-intuitive results for the optimization of
such mixers.These are
summarized below.

Figure
5.Simulation of convective diffusion (and
subsequent mixing rate) of protein denaturant from center stream (a-c).Model shows that the thinnest stream
(c) is not the fastest mixing condition. On the right (d) is a quantitative
(experimental) image of species concentration in the optimized mixer.

In the right-most flow field
above, a thin center stream results in low advection velocities and mixing
time is dominated by two-dimensional convective-diffusive transport in the
“arrow-head” shaped focusing region.On the right-most flow field, the
transport in the focusing region is dominated by advection and mixing time
is limited by diffusion in the spanwise direction (normal to streamlines)
from the relatively thick center stream.The fastest mixing time (for these conditions, less than 8 us) are for the
center, intermediate case.

A second counter-intuitive
result is that, for near-optimal conditions, denaturants with higher
diffusivities actually mix more slowly than denaturants with lower diffusivities.That is, mixing time increases with
diffusivity for near optimal conditions. This second counter-intuitive result
is due to the non-linear effects associated with convective diffusion in
the initial focusing region.Low diffusivity denaturants enable optimal mixer conditions with
thin, low center stream flow rates which have spanwise-diffusion-dominated
transport and minimize tmix.High diffusivity denaturants require relatively thick, high center
streams flow rates which exhibit substantial two-dimensional diffusion
transport in the initial focusing region.

Experimental Mixing

We use techniques such as dye
quenching and collapse of single stranded DNA to measure the mixing times of
our mixers.Figure 6a
(below-left) shows results of a dye quenching experiment with two different
mixer times.We have also
demonstrated feasibility of protein folding by measuring collapse and
folding rates of a well studied benchmark protein (below-right).

Figure 6. (a)
Results from fluorescence quenching experiments showing concentration of potassium
iodide versus time for our original mixer design and a shape-optimized
design.Mixing is complete in
approximately 7 ms for
the original design and 4 ms for our
newest optimized design. (b)Experiment measuring folding rates of ACBP.Signal indicates proximity of two fluorophores (FRET) attached to opposite ends of the
protein.A large signal
indicates the protein is in a folded state.Our measured kinetic rates at 90 ms and 8 ms agree well with
published results.By
increasing the detection channel width downstream of the focusing region we
are able to measure kinetics from under 10 ms to as long as 100 ms.

We have
optimized, designed, and characterized a microsecond microfluidic mixer
using hydrodynamic focusing, and have demonstrated as short as 4 ms mixing times with fluorescence quenching and collapse of
single-stranded DNA.Our mixer
has been used to measure the folding rates of the protein acyl-CoA-binding protein (ACBP) and chymotrypsin inhibitor-2 (CI2), and is currently being used to investigate cytochrome-c with UV and three-photon detection
techniques.